Nucleic acids encoding sugar transport proteins and methods of using same

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a sugar transport protein. The invention also relates to the construction of a chimeric gene encoding all or a portion of the sugar transport protein, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the sugar transport protein in a transformed host cell.

This application is a divisional of U.S. application Ser. No. 11/210,316filed Aug. 24, 2005, now U.S. Pat. No. 7,332,300 now granted, which isdivisional of U.S. application Ser. No. 10/051,902 filed Jan. 17, 2002,now granted as U.S. Pat. No. 7,189,531, which is a divisional of U.S.application Ser. No. 09/291,922, filed Apr. 14, 1999, now granted asU.S. Pat. No. 6,383,776, which claims the benefit of U.S. ProvisionalApplication No. 60/083,044, filed Apr. 24, 1998, the entire contents ofwhich are herein incorporated by reference.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingsugar transport proteins in plants and seeds.

BACKGROUND OF THE INVENTION

Sugar is one form of carbohydrate produced in photosynthesizing cells inmost higher plants and is the main form of transported carbon in mostannual field crops such as corn, rice, soybeans and wheat. As such itsmovement and concentration across various plant membranes is critical toplant growth and development. In addition sugar is the main form ofcarbon that moves into developing seeds of soybeans, rice, corn andwheat. This movement and concentration is accomplished by the action ofcarrier proteins that act to transport sugar against a concentrationgradient often by coupling sugar movement to the opposite vectoralmovement of a proton. Specific sugar carrier proteins from these cropplants could be manipulated in efforts to control carbon flux and thetiming and extent of sugar transport phenomena (e.g., grain fillduration) that are important factors in crop yield and quality.Accordingly, the availability of nucleic acid sequences encoding all ora portion of sugar transport proteins would facilitate studies to betterunderstand carbon flux and sugar transport in plants, provide genetictools for the manipulation of sugar transport, and provide a means tocontrol carbohydrate transport and distribution in plant cells.

SUMMARY OF THE INVENTION

The instant invention relates to isolated nucleic acid fragmentsencoding sugar transport proteins. Specifically, this invention concernsan isolated nucleic acid fragment encoding an Arabidopsis thaliana-likesugar transport protein or Beta vulgaris-like sugar transport protein.In addition, this invention relates to a nucleic acid fragment that iscomplementary to the nucleic acid fragment encoding an Arabidopsisthaliana-like sugar transport protein or Beta vulgaris-like sugartransport protein.

An additional embodiment of the instant invention pertains to apolypeptide encoding all or a substantial portion of a sugar transportprotein selected from the group consisting of Arabidopsis thaliana-likesugar transport protein and Beta vulgaris-like sugar transport protein.

In another embodiment, the instant invention relates to a chimeric geneencoding an Arabidopsis thaliana-like sugar transport protein or Betavulgaris-like sugar transport protein, or to a chimeric gene thatcomprises a nucleic acid fragment that is complementary to a nucleicacid fragment encoding an Arabidopsis thaliana-like sugar transportprotein or Beta vulgaris-like sugar transport protein, operably linkedto suitable regulatory sequences, wherein expression of the chimericgene results in production of levels of the encoded protein in atransformed host cell that is altered (i.e., increased or decreased)from the level produced in an untransformed host cell.

In a further embodiment, the instant invention concerns a transformedhost cell comprising in its genome a chimeric gene encoding anArabidopsis thaliana-like sugar transport protein or Beta vulgaris-likesugar transport protein, operably linked to suitable regulatorysequences. Expression of the chimeric gene results in production ofaltered levels of the encoded protein in the transformed host cell. Thetransformed host cell can be of eukaryotic or prokaryotic origin, andinclude cells derived from higher plants and microorganisms. Theinvention also includes transformed plants that arise from transformedhost cells of higher plants, and seeds derived from such transformedplants.

An additional embodiment of the instant invention concerns a method ofaltering the level of expression of an Arabidopsis thaliana-like sugartransport protein or Beta vulgaris-like sugar transport protein in atransformed host cell comprising: a) transforming a host cell with achimeric gene comprising a nucleic acid fragment encoding an Arabidopsisthaliana-like sugar transport protein or Beta vulgaris-like sugartransport protein; and b) growing the transformed host cell underconditions that are suitable for expression of the chimeric gene whereinexpression of the chimeric gene results in production of altered levelsof Arabidopsis thaliana-like sugar transport protein or Betavulgaris-like sugar transport protein in the transformed host cell.

An addition embodiment of the instant invention concerns a method forobtaining a nucleic acid fragment encoding all or a substantial portionof an amino acid sequence encoding an Arabidopsis thaliana-like sugartransport protein or Beta vulgaris-like sugar transport protein.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIGS. 1A, 1B, 1C, 1D, 1E, 1F and 1G show a comparison of the amino acidsequences set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14 and 16 with theArabidopsis thaliana-like sugar transport protein amino acid sequenceset forth in SEQ ID NO:29. Amino acid designations in small case lettersrepresent regions that are thought to be Arabidopsis thaliana-like sugartransport protein signatures.

FIGS. 2A, 2B, 2C and 2D show a comparison of the amino acid sequencesset forth in SEQ ID NOs:18, 20, 22, 24, 26 and 28 with the Betavulgaris-like sugar transport protein amino acid sequence set forth inSEQ ID NO:30.

The following sequence descriptions and Sequence Listing attached heretocomply with the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825.

SEQ ID NO:1 is the nucleotide sequence comprising a contig assembledfrom the cDNA inserts in clones p0032.crcba66r, p0097.cqran41r,cr1n.pk0143.h10, p0128.cpict38, p0106.cjlpm67r, cil1c.pk001.f21,p0072.comgi92r, p0114.cimm181r and p0002.cgevb73r encoding a cornArabidopsis thaliana-like sugar transport protein.

SEQ ID NO:2 is the deduced amino acid sequence of an Arabidopsisthaliana-like sugar transport protein derived from the nucleotidesequence of SEQ ID NO:1.

SEQ ID NO:3 is the nucleotide sequence comprising a contig assembledfrom the cDNA inserts in clones rlr12.pk0013.d11 and rds1c.pk007.n17encoding a portion of a rice Arabidopsis thaliana-like sugar transportprotein.

SEQ ID NO:4 is the deduced amino acid sequence of a portion of anArabidopsis thaliana-like sugar transport protein derived from thenucleotide sequence of SEQ ID NO:3.

SEQ ID NO:5 is the nucleotide sequence comprising a the entire cDNAinsert in clone rls6.pk0003.d5 encoding a portion of a rice Arabidopsisthaliana-like sugar transport protein.

SEQ ID NO:6 is the deduced amino acid sequence of an Arabidopsisthaliana-like sugar transport protein derived from the nucleotidesequence of SEQ ID NO:5.

SEQ ID NO:7 is the nucleotide sequence comprising a contig assembledfrom the cDNA inserts in clones sgs4c.pk005.c9, sfl1.pk0079.a4 andsdp3c.pk012.i1 encoding a soybean Arabidopsis thaliana-like sugartransport protein.

SEQ ID NO:8 is the deduced amino acid sequence of an Arabidopsisthaliana-like sugar transport protein derived from the nucleotidesequence of SEQ ID NO:7.

SEQ ID NO:9 is the nucleotide sequence comprising a portion of the cDNAinsert in clone ss1.pk0022.f1 encoding a portion of a soybeanArabidopsis thaliana-like sugar transport protein.

SEQ ID NO:10 is the deduced amino acid sequence of a portion of anArabidopsis thaliana-like sugar transport protein derived from thenucleotide sequence of SEQ ID NO:9.

SEQ ID NO:11 is the nucleotide sequence comprising a portion of the cDNAinsert in clone wlk8.pk0001.a12 encoding a portion of a wheatArabidopsis thaliana-like sugar transport protein.

SEQ ID NO:12 is the deduced amino acid sequence of a portion of anArabidopsis thaliana-like sugar transport protein derived from thenucleotide sequence of SEQ ID NO:11.

SEQ ID NO:13 is the nucleotide sequence comprising a contig assembledfrom the cDNA inserts in clones wlm96.pk043.e19 and wre1n.pk0062.g6encoding a portion of a wheat Arabidopsis thaliana-like sugar transportprotein.

SEQ ID NO:14 is the deduced amino acid sequence of a portion of anArabidopsis thaliana-like sugar transport protein derived from thenucleotide sequence of SEQ ID NO:13.

SEQ ID NO:15 is the nucleotide sequence comprising a portion of the cDNAinsert in clone wre1n.pk0006.b4 encoding a portion of a wheatArabidopsis thaliana-like sugar transport protein.

SEQ ID NO:16 is the deduced amino acid sequence of a portion of anArabidopsis thaliana-like sugar transport protein derived from thenucleotide sequence of SEQ ID NO:15.

SEQ ID NO:17 is the nucleotide sequence comprising a portion of the cDNAinsert in clone cc1.mn0002.h1 encoding a portion of a corn Betavulgaris-like sugar transport protein.

SEQ ID NO:18 is the deduced amino acid sequence of a portion of a Betavulgaris-like sugar transport protein derived from the nucleotidesequence of SEQ ID NO: 17.

SEQ ID NO: 19 is the nucleotide sequence comprising the entire cDNAinsert in clone cepe7.pk0018.g3 encoding a corn Beta vulgaris-like sugartransport protein.

SEQ ID NO:20 is the deduced amino acid sequence of a Beta vulgaris-likesugar transport protein derived from the nucleotide sequence of SEQ IDNO:19.

SEQ ID NO:21 is the nucleotide sequence comprising a contig assembledfrom the cDNA inserts in clones rlr6.pk0005.b10, rl0n.pk102.p24 andrl0n.pk107.p2 encoding a rice Beta vulgaris-like sugar transportprotein.

SEQ ID NO:22 is the deduced amino acid sequence of a Beta vulgaris-likesugar transport protein derived from the nucleotide sequence of SEQ IDNO:21.

SEQ ID NO:23 is the nucleotide sequence comprising a contig assembledfrom the cDNA inserts in clones sr1.pk0061.g8, sfl1.pk0058.h12,sgs2c.pk004.o17 and sre.pk0032.h6 encoding a soybean Beta vulgaris-likesugar transport protein.

SEQ ID NO:24 is the deduced amino acid sequence of a Beta vulgaris-likesugar transport protein derived from the nucleotide sequence of SEQ IDNO:23.

SEQ ID NO:25 is the nucleotide sequence comprising the entire cDNAinsert in clone wlk8.pk0001.a11 encoding a wheat Beta vulgaris-likesugar transport protein.

SEQ ID NO:26 is the deduced amino acid sequence of a Beta vulgaris-likesugar transport protein derived from the nucleotide sequence of SEQ IDNO:25.

SEQ ID NO:27 is the nucleotide sequence comprising the entire cDNAinsert in clone wlm1.pk0012.h1 encoding a wheat Beta vulgaris-like sugartransport protein.

SEQ ID NO:28 is the deduced amino acid sequence of a Beta vulgaris-likesugar transport protein derived from the nucleotide sequence of SEQ IDNO:28.

SEQ ID NO:29 is the amino acid sequence of an Arabidopsis thaliana (NCBIIdentification No. gi 3080420) sugar transport protein.

SEQ ID NO:30 is the amino acid sequence of a Beta vulgaris (NCBIIdentification No. gi 1778093) sugar transport protein.

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA. As used herein,“contig” refers to an assemblage of overlapping nucleic acid sequencesto form one contiguous nucleotide sequence. For example, several DNAsequences can be compared and aligned to identify common or overlappingregions. The individual sequences can then be assembled into a singlecontiguous nucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence.

“Substantially similar” also refers to nucleic acid fragments whereinchanges in one or more nucleotide bases does not affect the ability ofthe nucleic acid fragment to mediate alteration of gene expression byantisense or co-suppression technology. “Substantially similar” alsorefers to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate alteration of geneexpression by antisense or co-suppression technology or alteration ofthe functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary sequences.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a gene which resultin the production of a chemically equivalent amino acid at a given site,but do not effect the functional properties of the encoded protein, arewell known in the art. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine, can also beexpected to produce a functionally equivalent product. Nucleotidechanges which result in alteration of the N-terminal and C-terminalportions of the protein molecule would also not be expected to alter theactivity of the protein. Each of the proposed modifications is wellwithin the routine skill in the art, as is determination of retention ofbiological activity of the encoded products.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize, under stringent conditions(0.1×SSC, 0.1% SDS, 65° C.), with the nucleic acid fragments disclosedherein.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent similarity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Preferred are those nucleic acid fragments whose nucleotidesequences encode amino acid sequences that are 90% similar to the aminoacid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% similar to theamino acid sequences reported herein. Sequence alignments and percentsimilarity calculations were performed using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins, D. G. and Sharp, P. M. (1989) CABIOS.5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTHPENALTY=10) (hereafter, Clustal algorithm). Default parameters forpairwise alignments using the Clustal method were KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to afford putative identification of thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410).In general, a sequence of ten or more contiguous amino acids or thirtyor more nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches partial or complete amino acid andnucleotide sequences encoding one or more particular plant proteins. Theskilled artisan, having the benefit of the sequences as reported herein,may now use all or a substantial portion of the disclosed sequences forpurposes known to those skilled in this art. Accordingly, the instantinvention comprises the complete sequences as reported in theaccompanying Sequence Listing, as well as substantial portions of thosesequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment that encodes all or a substantialportion of the amino acid sequence encoding the Arabidopsisthaliana-like sugar transport proteins or Beta vulgaris-like sugartransport proteins as set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26 and 28. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing agene for improved expression in a host cell, it is desirable to designthe gene such that its frequency of codon usage approaches the frequencyof preferred codon usage of the host cell.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, (1989) Biochemistry of Plants 15: 1-82. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of differentlengths may have identical promoter activity.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D. (1995)Molecular Biotechnology 3:225).

The “3′ non-coding sequences” refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065,incorporated herein by reference). The complementarity of an antisenseRNA may be with any part of the specific gene transcript, i.e., at the5′ non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence. “Functional RNA” refers to sense RNA, antisense RNA, ribozymeRNA, or other RNA that may not be translated but yet has an effect oncellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels, J.J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If theprotein is to be directed to a vacuole, a vacuolar targeting signal(supra) can further be added, or if to the endoplasmic reticulum, anendoplasmic reticulum retention signal (supra) may be added. If theprotein is to be directed to the nucleus, any signal peptide presentshould be removed and instead a nuclear localization signal included(Raikhel (1992) Plant Phys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of several sugartransport proteins have been isolated and identified by comparison ofrandom plant cDNA sequences to public databases containing nucleotideand protein sequences using the BLAST algorithms well known to thoseskilled in the art. Table 1 lists the proteins that are describedherein, and the designation of the cDNA clones that comprise the nucleicacid fragments encoding these proteins.

TABLE 1 Sugar Transport Proteins Enzyme Clone Plant Sugar TransportProtein p0032.crcba66r Corn (Arabidopsis-like) p0097.cqran41r Corncr1n.pk0143.h10 Corn p0128.cpict38 Corn p0106.cjlpm67r Corncil1c.pk001.f21 Corn p0072.comgi92r Corn p0114.cimm181r Cornp0002.cgevb73r Corn rds1c.pk007.n17 Rice rlr12.pk0013.d11 Ricerls6.pk0003.d5 Rice sgs4c.pk005.c9 Soybean sfl1.pk0079.a4 Soybeansdp3c.pk012.i1 Soybean ss1.pk0022.f1 Soybean wlk8.pk0001.a12 Wheatwlm96.pk043.e19 Wheat wre1n.pk0062.g6 Wheat wre1n.pk0006.b4 Wheat SugarTransport Protein cc1.mn0002.h1 Corn (Beta vulgaris-like)cepe7.pk0018.g3 Corn rlr6.pk0005.b10 Rice rl0n.pk102.p24 Ricerl0n.pk107.p2 Rice sr1.pk0061.g8 Soybean sfl1.pk0058.h12 Soybeansgs2c.pk004.o17 Soybean sre.pk0032.h6 Soybean wlk8.pk0001.a11 Wheatwlm1.pk0012.h1 Wheat

The nucleic acid fragments of the instant invention may be used toisolate cDNAs and genes encoding homologous proteins from the same orother plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other Arabidopsis thaliana-like sugartransport proteins or Beta vulgaris-like sugar transport proteins,either as cDNAs or genomic DNAs, could be isolated directly by using allor a portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al., (1988) PNAS USA 85:8998) to generate cDNAs byusing PCR to amplify copies of the region between a single point in thetranscript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′directions can be designed from the instant sequences. Usingcommercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or5′ cDNA fragments can be isolated (Ohara et al., (1989) PNAS USA86:5673; Loh et al., (1989) Science 243:217). Products generated by the3′ and 5′ RACE procedures can be combined to generate full-length cDNAs(Frohman, M. A. and Martin, G. R., (1989) Techniques 1:165).

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner, R. A. (1984) Adv.Immunol. 36:1; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed Arabidopsisthaliana-like sugar transport proteins or Beta vulgaris-like sugartransport proteins are present at higher or lower levels than normal orin cell types or developmental stages in which they are not normallyfound. This would have the effect of altering the level of sugartransport in those cells.

Overexpression of the Arabidopsis thaliana-like sugar transport proteinsor Beta vulgaris-like sugar transport proteins of the instant inventionmay be accomplished by first constructing a chimeric gene in which thecoding region is operably linked to a promoter capable of directingexpression of a gene in the desired tissues at the desired stage ofdevelopment. For reasons of convenience, the chimeric gene may comprisepromoter sequences and translation leader sequences derived from thesame genes. 3′ Non-coding sequences encoding transcription terminationsignals may also be provided. The instant chimeric gene may alsocomprise one or more introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can thenconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instant sugartransport proteins to different cellular compartments, or to facilitateits secretion from the cell. It is thus envisioned that the chimericgene described above may be further supplemented by altering the codingsequence to encode Arabidopsis thaliana-like sugar transport proteins orBeta vulgaris-like sugar transport proteins with appropriateintracellular targeting sequences such as transit sequences (Keegstra,K. (1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels, J. J., (1991) Ann. Rev.Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel, N. (1992) Plant Phys. 100:1627-1632) added and/or withtargeting sequences that are already present removed. While thereferences cited give examples of each of these, the list is notexhaustive and more targeting signals of utility may be discovered inthe future.

It may also be desirable to reduce or eliminate expression of genesencoding Arabidopsis thaliana-like sugar transport proteins or Betavulgaris-like sugar transport proteins in plants for some applications.In order to accomplish this, a chimeric gene designed for co-suppressionof the instant sugar transport proteins can be constructed by linking agene or gene fragment encoding an Arabidopsis thaliana-like sugartransport protein or Beta vulgaris-like sugar transport protein to plantpromoter sequences. Alternatively, a chimeric gene designed to expressantisense RNA for all or part of the instant nucleic acid fragment canbe constructed by linking the gene or gene fragment in reverseorientation to plant promoter sequences. Either the co-suppression orantisense chimeric genes could be introduced into plants viatransformation wherein expression of the corresponding endogenous genesare reduced or eliminated.

The instant Arabidopsis thaliana-like sugar transport proteins or Betavulgaris-like sugar transport proteins (or portions thereof) may beproduced in heterologous host cells, particularly in the cells ofmicrobial hosts, and can be used to prepare antibodies to the theseproteins by methods well known to those skilled in the art. Theantibodies are useful for detecting Arabidopsis thaliana-like sugartransport proteins or Beta vulgaris-like sugar transport proteins insitu in cells or in vitro in cell extracts. Preferred heterologous hostcells for production of the instant sugar transport proteins aremicrobial hosts. Microbial expression systems and expression vectorscontaining regulatory sequences that direct high level expression offoreign proteins are well known to those skilled in the art. Any ofthese could be used to construct a chimeric gene for production of theinstant Arabidopsis thaliana-like sugar transport proteins or Betavulgaris-like sugar transport proteins. This chimeric gene could then beintroduced into appropriate microorganisms via transformation to providehigh level expression of the encoded sugar transport protein. An exampleof a vector for high level expression of the instant Arabidopsisthaliana-like sugar transport proteins or Beta vulgaris-like sugartransport proteins in a bacterial host is provided (Example 7).

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al., (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein, D. et al., (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in R. Bernatzky, R. and Tanksley, S. D. (1986)Plant Mol. Biol. Reporter 4(1):37-41. Numerous publications describegenetic mapping of specific cDNA clones using the methodology outlinedabove or variations thereof. For example, F2 intercross populations,backcross populations, randomly mated populations, near isogenic lines,and other sets of individuals may be used for mapping. Suchmethodologies are well known to those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel, J. D., et al., In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask, B. J. (1991) Trends Genet.7:149-154). Although current methods of FISH mapping favor use of largeclones (several to several hundred KB; see Laan, M. et al. (1995) GenomeResearch 5:13-20), improvements in sensitivity may allow performance ofFISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian, H.H. (1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism ofPCR-amplified fragments (CAPS; Sheffield, V. C. et al. (1993) Genomics16:325-332), allele-specific ligation (Landegren, U. et al. (1988)Science 241:1077-1080), nucleotide extension reactions (Sokolov, B. P.(1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter, M.A. et al. (1997) Nature Genetics 7:22-28) and Happy Mapping (Dear, P. H.and Cook, P. R. (1989) Nucleic Acid Res. 17:6795-6807). For thesemethods, the sequence of a nucleic acid fragment is used to design andproduce primer pairs for use in the amplification reaction or in primerextension reactions. The design of such primers is well known to thoseskilled in the art. In methods employing PCR-based genetic mapping, itmay be necessary to identify DNA sequence differences between theparents of the mapping cross in the region corresponding to the instantnucleic acid sequence. This, however, is generally not necessary formapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer, (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al., (1995)Proc. Natl. Acad. Sci USA 92:8149; Bensen et al., (1995) Plant Cell7:75). The latter approach may be accomplished in two ways. First, shortsegments of the instant nucleic acid fragments may be used in polymerasechain reaction protocols in conjunction with a mutation tag sequenceprimer on DNAs prepared from a population of plants in which Mutatortransposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the Arabidopsis thaliana-likesugar transport protein or Beta vulgaris-like sugar transport protein.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding anArabidopsis thaliana-like sugar transport protein or Beta vulgaris-likesugar transport protein can be identified and obtained. This mutantplant can then be used to determine or confirm the natural function ofthe Arabidopsis thaliana-like sugar transport protein or Betavulgaris-like sugar transport protein gene product.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various corn, rice, soybean andwheat tissues were prepared. The characteristics of the libraries aredescribed below.

TABLE 2 cDNA Libraries from Corn, Rice, Soybean and Wheat Library TissueClone cc1 Corn (Zea mays L.) callus stage 1** cc1.mn0002.h1 Cepe7 Corn(Zea mays L.) epicotyl from 7 day old etiolated cepe7.pk0018.g3 seedlingcil1c Corn (Zea mays L.) pooled immature leaf tissue at V4,cil1c.pk001.f21 V6 and V8** cr1n Corn (Zea mays L.) root from 7 dayseedlings grown in cr1n.pk0143.h10 light* p0002 Corn (Zea mays L.)tassel: premeiotic > early uninucleate p0002.cgevb73r p0032 Corn (Zeamays L.) regenernerating callus, 10 and 14 days p0032.crcba66r afterauxin removal. p0072 Corn (Zea mays L.) 14 days after planting etiolatedp0072.comgi92r seedling: mesocotyl p0097 Corn (Zea mays L.) V9, 7 cmwhorl section after p0097.cqran41r application of European Corn Borerp0106 Corn (Zea mays L.) 5 days after pollenation whole kernels*p0106.cjlpm67r p0114 Corn (Zea mays L.) intercalary meristem ofexpanding p0114.cimm181r internodes 5-9 at V10 stage* p0128 Corn (Zeamays L.) pooled primary and secondary p0128.cpict38 immature ear Rds1cRice (Oryza sativa, YM) developing seeds rds1c.pk007.n17 rlr6 Rice(Oryza sativa L.) leaf (15 days after germination) rlr6.pk0005.b10 6 hrsafter infection of Magaporthe grisea strain 4360-R-62 (AVR2-YAMO);Resistant rl0n Rice (Oryza sativa L.) 15 day leaf* rl0n.pk102.p24rl0n.pk107.p2 rlr12 Rice (Oryza sativa L.) leaf, 15 days aftergermination, rlr12.pk0013.d11 12 hours after infection of Magaporthegrisea strain 4360-R-62 (AVR2-YAMO); Resistant rls6 Rice (Oryza sativaL.) leaf, 15 days after germination, rls6.pk0003.d5 6 hrs afterinfection of Magaporthe grisea strain 4360-R-67 (avr2-yamo); Susceptiblesdp3c Soybean (Glycine max L.) developing pods 8-9 mm sdp3c.pk012.i1sfl1 Soybean (Glycine max L.) immature flower sfl1.pk0079.a4sfl1.pk0058.h12 sgs2c Soybean (Glycine max L.) seeds 14 hrs aftergermination sgs2c.pk004.o17 sgs4c Soybean (Glycine max L.) seeds 2 daysafter germination sgs4c.pk005.c9 sr1 Soybean (Glycine max L.) rootlibrary sr1.pk0061.g8 Sre Soybean (Glycine max L.) root elongationsre.pk0032.h6 ss1 Soybean (Glycine max L.) seedling 5-10 dayss1.pk0022.f1 wlk8 Wheat (Triticum aestivum L.) seedlings 8 hr afterwlk8.pk0001.a11 treatment with fungicide*** wlk8.pk0001.a12 wlm1 Wheat(Triticum aestivum L.) seedlings 1 hr after wlm1.pk0012.h1 inoculationwith Erysiphe graminis f. sp tritici wlm96 Wheat (Triticum aestivum L.)seedlings 96 hr after wlm96.pk043.e19 inoculation w/E. graminis wre1nWheat (Triticum aestivum L.) root; 7 day old etiolated wre1n.pk0006.b4seedling* wre1n.pk0062.g6 *These libraries were normalized essentiallyas described in U.S. Pat. No. 5,482,845 **V4, V6 and V8 refer to stagesof corn growth. The descriptions can be found in “How a Corn PlantDevelops” Special Report No. 48, Iowa State University of Science andTechnology Cooperative Extension Service Ames, Iowa, Reprinted February1996. ***Application of 6-iodo-2-propoxy-3-propyl-4(3H)-quinazolinone;synthesis and methods of using this compound are described in USSN08/545,827, incorporated herein by reference.

cDNA libraries were prepared in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).Conversion of the Uni-ZAP™ XR libraries into plasmid libraries wasaccomplished according to the protocol provided by Stratagene. Uponconversion, cDNA inserts were contained in the plasmid vectorpBluescript. cDNA inserts from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids were amplified viapolymerase chain reaction using primers specific for vector sequencesflanking the inserted cDNA sequences or plasmid DNA was prepared fromcultured bacterial cells. Amplified insert DNAs or plasmid DNAs weresequenced in dye-primer sequencing reactions to generate partial cDNAsequences (expressed sequence tags or “ESTs”; see Adams, M. D. et al.,(1991) Science 252:1651). The resulting ESTs were analyzed using aPerkin Elmer Model 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

ESTs encoding sugar transport proteins were identified by conductingBLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al.,(1993) J. Mol. Biol. 215:403-410) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish, W. and States, D. J. (1993) Nature Genetics 3:266-272 andAltschul, Stephen F., et al. (1997) Nucleic Acids Res. 25:3389-3402)provided by the NCBI. For convenience, the P-value (probability) ofobserving a match of a cDNA sequence to a sequence contained in thesearched databases merely by chance as calculated by BLAST are reportedherein as “pLog” values, which represent the negative of the logarithmof the reported P-value. Accordingly, the greater the pLog value, thegreater the likelihood that the cDNA sequence and the BLAST “hit”represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Arabidopsisthaliana-like Sugar Transport Proteins

The BLASTX search using the EST sequences from several corn, rice,soybean and wheat clones revealed similarity of the proteins encoded bythe cDNAs to a sugar transport protein from Arabidopsis thaliana (NCBIIdentifier No. gi 3080420). In the process of comparing the ESTs it wasfound that many of the clones had overlapping regions of homology. Usingthis homology it was possible to align the ESTs and assemble severalcontigs encoding unique corn, rice, soybean and wheat sugar transportproteins. The individual clones and the composition of each assembledcontig are shown in Table 3. The BLAST results for each of the contigsand individual ESTs and are also shown in Table 3:

TABLE 3 BLAST Results for Clones Encoding Polypeptides Homologous toArabidopsis thaliana Sugar Transport Protein Clone BLAST pLog ScoreContig composed of clones: >250.00 p0032.crcba66r p0097.cqran41rcr1n.pk0143.h10 p0128.cpict38 p0106.cjlpm67r cil1c.pk001.f21p0072.comgi92r p0114.cimm181r p0002.cgevb73r Contig composed of clones:27.70 rlr12.pk0013.d11 rds1c.pk007.n17 rls6.pk0003.d5 54.00 Contigcomposed of clones: >250.00 sgs4c.pk005.c9 sfl1.pk0079.a4 sdp3c.pk012.i1ss1.pk0022.f1 >250.00 wlk8.pk0001.a12 21.30 Contig composed of clones:149.00 Wlm96.pk043.e19 wre1n.pk0062.g6 wre1n.pk0006.b4 117.00

The sequence of the corn contig composed of clones p0032.crcba66r,p0097.cqran41r, cr1n.pk0143.h10, p0128.cpict38, p0106.cjlpm67r,cil1c.pk001.f21, p0072.comgi92r, p0114.cimm181r and p0002.cgevb73r isshown in SEQ ID NO:1; the deduced amino acid sequence of this contig,which represents 100% of the protein, is shown in SEQ ID NO:2. Acalculation of the percent similarity of the amino acid sequence setforth in SEQ ID NO:2 and the Arabidopsis thaliana sequence (using theClustal algorithm) revealed that the protein encoded by SEQ ID NO:2 is66% similar to the Arabidopsis thaliana sugar transport protein.

The sequence of the rice contig composed of clones rlr12.pk0013.d11 andrds1c.pk007.n17 is shown in SEQ ID NO:3; the deduced amino acid sequenceof this contig, which represents 9% of the protein (N-terminal region),is shown in SEQ ID NO:4. A calculation of the percent similarity of theamino acid sequence set forth in SEQ ID NO:4 and the Arabidopsisthaliana sequence (using the Clustal algorithm) revealed that theprotein encoded by SEQ ID NO:2 is 86% similar to the Arabidopsisthaliana sugar transport protein.

The sequence of the entire cDNA insert from clone rls6.pk0003.d5 isshown in SEQ ID NO:5; the deduced amino acid sequence of this cDNA,which represents 18% of the of the protein (C-terminal region), is shownin SEQ ID NO:6. A calculation of the percent similarity of the aminoacid sequence set forth in SEQ ID NO:6 and the Arabidopsis thalianasequence (using the Clustal algorithm) revealed that the protein encodedby SEQ ID NO:6 is 74% similar to the Arabidopsis thaliana sugartransport protein.

The sequence of the soybean contig composed of clones sgs4c.pk005.c9,sfl1.pk0079.a4 and sdp3c.pk012.i1 is shown in SEQ ID NO:7; the deducedamino acid sequence of this contig, which represents 100% of theprotein, is shown in SEQ ID NO:8. A calculation of the percentsimilarity of the amino acid sequence set forth in SEQ ID NO:8 and theArabidopsis thaliana sequence (using the Clustal algorithm) revealedthat the protein encoded by SEQ ID NO:8 is 68% similar to theArabidopsis thaliana sugar transport protein.

The sequence of a portion of the cDNA insert from clone ss1.pk0022.f1 isshown in SEQ ID NO:9; the deduced amino acid sequence of this cDNA,which represents 66% of the of the protein (C-terminal region), is shownin SEQ ID NO:10. A calculation of the percent similarity of the aminoacid sequence set forth in SEQ ID NO:10 and the Arabidopsis thalianasequence (using the Clustal algorithm) revealed that the protein encodedby SEQ ID NO:10 is 66% similar to the Arabidopsis thaliana sugartransport protein.

The sequence of a portion of the cDNA insert from clone wlk8.pk0001.a12is shown in SEQ ID NO:11; the deduced amino acid sequence of this cDNA,which represents 7% of the of the protein (N-terminal region), is shownin SEQ ID NO:12. A calculation of the percent similarity of the aminoacid sequence set forth in SEQ ID NO:12 and the Arabidopsis thalianasequence (using the Clustal algorithm) revealed that the protein encodedby SEQ ID NO:12 is 88% similar to the Arabidopsis thaliana sugartransport protein.

The sequence of the wheat contig composed of clones wlm96.pk043.e19 andwre1n.pk0062.g6 is shown in SEQ ID NO:13; the deduced amino acidsequence of this contig, which represents 45% of the protein (C-terminalregion), is shown in SEQ ID NO:14. A calculation of the percentsimilarity of the amino acid sequence set forth in SEQ ID NO:14 and theArabidopsis thaliana sequence (using the Clustal algorithm) revealedthat the protein encoded by SEQ ID NO:14 is 65% similar to theArabidopsis thaliana sugar transport protein.

The sequence of a portion of the cDNA insert from clone wre1n.pk0006.b4is shown in SEQ ID NO:15; the deduced amino acid sequence of this cDNA,which represents 31% of the of the protein (C-terminal region), is shownin SEQ ID NO:16. A calculation of the percent similarity of the aminoacid sequence set forth in SEQ ID NO:16 and the Arabidopsis thalianasequence (using the Clustal algorithm) revealed that the protein encodedby SEQ ID NO:16 is 76% similar to the Arabidopsis thaliana sugartransport protein.

FIGS. 1A, 1B, 1C, 1D, 1E, 1F and 1G present an alignment of the aminoacid sequence set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14 and 16 withthe Arabidopsis thaliana-like sugar transport protein amino acidsequence, SEQ ID NO:29. Alignments were performed using the Clustalalgorithm. The percent similarity between the corn, rice, soybean andwheat acid sequences was calculated to range between 16% to 89% usingthe Clustal algorithm.

BLAST scores and probabilities indicate that the instant nucleic acidfragments encode portions of sugar transport proteins. These sequencesrepresent the first corn, rice, soybean and wheat sequences encodingArabidopsis thaliana-like sugar transport proteins.

Example 4 Characterization of cDNA Clones Encoding Beta vulgaris-likeSugar Transport Proteins

The BLASTX search using the EST sequences from several corn, rice,soybean and wheat clones revealed similarity of the proteins encoded bythe cDNAs to a sugar transport protein from Beta vulgaris (NCBIIdentifier No. gi 1778093). In the process of comparing the ESTs it wasfound that several of the rice and soybean clones had overlappingregions of homology. Using this homology it was possible to align theESTs and assemble two contigs encoding unique rice and soybean B.vulgaris-like sugar transport proteins. The individual clones and theassembled composition of each contig are shown in Table 4. The BLASTresults for each of the contigs and individual ESTs and are also shownin Table 4:

TABLE 4 BLAST Results for Clones Encoding Polypeptides Homologous toBeta vulgaris Sugar Transport Protein Clone BLAST pLog Scorecc1.mn0002.h1 53.70 cepe7.pk0018.g3 164.00 Contig composed ofclones: >250.00 rlr6.pk0005.b10 rl0n.pk102.p24 rl0n.pk107.p2 Contigcomposed of clones: >250.00 sr1.pk0061.g8 sfl1.pk0058.h12sgs2c.pk004.o17 sre.pk0032.h6 wlk8.pk0001.a11 >250.00 wlm1.pk0012.h1>250.00

The sequence of a portion of the cDNA insert from clone cc1.mn0002.h1 isshown in SEQ ID NO:17; the deduced amino acid sequence of this cDNA,which represents 31% of the of the protein (N-terminal region), is shownin SEQ ID NO:18. A calculation of the percent similarity of the aminoacid sequence set forth in SEQ ID NO:18 and the Beta vulgaris sequence(using the Clustal algorithm) revealed that the protein encoded by SEQID NO:18 is 65% similar to the Beta vulgaris sugar transport protein.

The sequence of the entire cDNA insert from clone cepe7.pk0018.g3 isshown in SEQ ID NO:19; the deduced amino acid sequence of this cDNA,which represents 100% of the of the protein, is shown in SEQ ID NO:20. Acalculation of the percent similarity of the amino acid sequence setforth in SEQ ID NO:20 and the Beta vulgaris sequence (using the Clustalalgorithm) revealed that the protein encoded by SEQ ID NO:20 is 57%similar to the Beta vulgaris sugar transport protein.

The sequence of the rice contig composed of clones rlr6.pk0005.b10,rl0n.pk102.p24 and rl0n.pk107.p2 is shown in SEQ ID NO:21; the deducedamino acid sequence of this contig, which represents 100% of theprotein, is shown in SEQ ID NO:22. A calculation of the percentsimilarity of the amino acid sequence set forth in SEQ ID NO:22 and theBeta vulgaris sequence (using the Clustal algorithm) revealed that theprotein encoded by SEQ ID NO:22 is 61% similar to the Beta vulgarissugar transport protein.

The sequence of the soybean contig composed of clones sr1.pk0061.g8,sfl1.pk0058.h12, sgs2c.pk004.o17 and sre.pk0032.h6 is shown in SEQ IDNO:23; the deduced amino acid sequence of this contig, which represents100% of the protein, is shown in SEQ ID NO:24. A calculation of thepercent similarity of the amino acid sequence set forth in SEQ ID NO:24and the Beta vulgaris sequence (using the Clustal algorithm) revealedthat the protein encoded by SEQ ID NO:23 is 66% similar to the Betavulgaris sugar transport protein.

The sequence of the entire cDNA insert from clone wlk8.pk0001.a 11 isshown in SEQ ID NO:25; the deduced amino acid sequence of this cDNA,which represents 100% of the of the protein, is shown in SEQ ID NO:26. Acalculation of the percent similarity of the amino acid sequence setforth in SEQ ID NO:26 and the Beta vulgaris sequence (using the Clustalalgorithm) revealed that the protein encoded by SEQ ID NO:26 is 61%similar to the Beta vulgaris sugar transport protein.

The sequence of the entire cDNA insert from clone wlm1.pk0012.h1 isshown in SEQ ID NO:27; the deduced amino acid sequence of this cDNA,which represents 100% of the of the protein, is shown in SEQ ID NO:28. Acalculation of the percent similarity of the amino acid sequence setforth in SEQ ID NO:28 and the Beta vulgaris sequence (using the Clustalalgorithm) revealed that the protein encoded by SEQ ID NO:28 is 56%similar to the Beta vulgaris sugar transport protein.

FIGS. 2A, 2B, 2C and 2D present an alignment of the amino acid sequenceset forth in SEQ ID NOs:18, 20, 22, 24, 26 and 28 with the Betavulgaris-like sugar transport protein amino acid sequence, SEQ ID NO:30.Alignments were performed using the Clustal algorithm. The percentsimilarity between the corn, rice, soybean and wheat acid sequences wascalculated to range between 43% to 81% using the Clustal algorithm.

BLAST scores and probabilities indicate that the instant nucleic acidfragments encode portions of sugar transport proteins. These sequencesrepresent the first corn, rice, soybean and wheat sequences encodingBeta vulgaris-like sugar transport proteins.

Example 5 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding sugar transport protein insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366, date of deposit Dec. 15, 1995.The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoterfragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI fragmentfrom the 3′ end of the maize 10 kD zein gene in the vector pGem9Zf(+)(Promega). Vector and insert DNA can be ligated at 15° C. overnight,essentially as described (Maniatis). The ligated DNA may then be used totransform E. coli XL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene).Bacterial transformants can be screened by restriction enzyme digestionof plasmid DNA and limited nucleotide sequence analysis using thedideoxy chain termination method (Sequenase™ DNA Sequencing Kit; U.S.Biochemical). The resulting plasmid construct would comprise a chimericgene encoding, in the 5′ to 3′ direction, the maize 27 kD zein promoter,a cDNA fragment encoding a sugar transport protein, and the 10 kD zein3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al., (1975) Sci. Sin. Peking 18:659-668). The embryos are keptin the dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al., (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al., (1990) Bio/Technology 8:833-839).

Example 6 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant sugar transport proteins in transformed soybean. Thephaseolin cassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising a sequence encoding a sugar transport protein. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Kline et al. (1987) Nature (London)327:70, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the sugar transport protein and thephaseolin 3′ region can be isolated as a restriction fragment. Thisfragment can then be inserted into a unique restriction site of thevector carrying the marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant sugar transport proteins can be insertedinto the T7 E. coli expression vector pBT430. This vector is aderivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) whichemploys the bacteriophage T7 RNA polymerase/T7 promoter system. PlasmidpBT430 was constructed by first destroying the EcoR I and Hind III sitesin pET-3a at their original positions. An oligonucleotide adaptorcontaining EcoR I and Hind III sites was inserted at the BamH I site ofpET-3a. This created pET-3aM with additional unique cloning sites forinsertion of genes into the expression vector. Then, the Nde I site atthe position of translation initiation was converted to an Nco I siteusing oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aMin this region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the sugar transport protein are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride) at pH 8.0containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A smallamount of 1 mm glass beads can be added and the mixture sonicated 3times for about 5 seconds each time with a microprobe sonicator. Themixture is centrifuged and the protein concentration of the supernatantdetermined. One μg of protein from the soluble fraction of the culturecan be separated by SDS-polyacrylamide gel electrophoresis. Gels can beobserved for protein bands migrating at the expected molecular weight.

1. An isolated nucleic acid comprising: (a) a nucleotide sequenceencoding a polypeptide having sugar transport protein activity, whereinsaid polypeptide is at least 95% identical to SEQ ID NO:22; or (b) thefull complement of the nucleotide sequence of (a).
 2. The isolatednucleic acid of claim 1, said nucleic acid comprises the nucleotidesequence of SEQ ID NO:21.
 3. A recombinant DNA construct comprising theisolated nucleic acid of claim 1 operably linked to a regulatorysequence.
 4. A vector comprising the isolated nucleic acid of claim 1.5. An isolated cell transformed with the recombinant DNA construct ofclaim
 3. 6. A method for increased production of a sugar transportprotein comprising: transforming a host cell with a chimeric genecomprising the nucleic acid of claim 1.